Project Summary/Abstract ?-adrenergic receptors (?ARs) play a central role in controlling the strength and frequency of cardiac contraction, and elicit Gs-linked cAMP generation. Disturbances of ?AR signaling have been implicated in many pathological conditions such as hypertension and heart failure. Upon agonist-induced activation, ?ARs are subject to a functional desensitization and endocytic removal from the plasma membrane (PM). Once inside the cells, the receptors were previously thought to be functionally inactive. Combining sophisticated imaging platforms with recently developed conformational biosensors, Dr. Irannejad directly probed activation of ?2AR and its cognate Gs protein and discovered that active ?2AR-G?s complex is not restricted to the PM and is also present at endosomes. Her long-term goal as an independent biomedical researcher is to understand the functional consequence of the ?AR endosomal signaling in regulating heart function. With this award, Dr. Irannejad will examine the role of endocytosis in ?ARs signaling in cardiomyocytes. This R99/R00 award will allow her to achieve the following career goal: 1) additional training in optical microscopy, 2) additional training in cardiomyocytes biology 3) develop skills to perform proteomic studies of protein complexes, 4) develop the communication, mentoring, grant-writing, and laboratory management skills necessary to become and successful, independent biomedical researcher. The mentored phase of this research will be carried out at the University of California, San Francisco under the guidance of Dr. Mark von Zastrow. During the mentored phase, Dr. Irannejad will use the conformational biosensors in cardiomyocytes to indentify compartmentalized ?ARs signaling (Aim1). Next, she will elucidate the role of endocytosis in signaling and contraction rate mediated by ?1AR versus ?2AR activation in cardiomyocytes (Aim 2). Dr. Shaun Coughlin (UCSF) director of CVRI at UCSF and an expert in cardiovascular biology will serve as co-mentor for this Aim. To indentify active ?2AR-Gs interactomes at the endosome (Aim 3), Dr. Irannejad will begin using proteomic approach. Dr. Nevan Krogan (UCSF), experts in proteomic studies of protein complexes will serve as collaborator and advisor. During the independent phase, Dr. Irannejad will continue to focus on Aim 2 and 3, characterize the dynamics of ?ARs-Gs mediated signaling, understand the functional consequence of the ?2AR endosomal signaling, and identifying ?2AR-Gs interactomes at the endosome. This award will enable Dr. Irannejad to elucidate the ?ARs compartmentalize signaling and its functional importance in regulating heart function and potentially developing new and better drugs for the treatment of pathological cardiac function.